Off-target capture reduction in sequencing techniques

ABSTRACT

Presented herein are methods and compositions for enhancing specific enrichment of target sequences in a nucleic acid library. Off-target hybridization probes may be used to reduce binding and/or capture of off-target regions of a nucleic acid library in a targeted sequencing workflow. The off-target hybridization probes may be specific for locations known to generate off-target sequencing reads for a particular set of hybridization probes.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/238,411, entitled “DATA-GUIDED DESIGN OF HYBRID CAPTUREOFF-TARGET REDUCERS” and filed Oct. 7, 2015, the disclosure of which isincorporated herein by reference for all purposes.

BACKGROUND

The present disclosure relates generally to the field of nucleic acidsequencing techniques. More particularly, the disclosure relates totechniques for enriching target capture and reducing off-target captureof nucleic acids to be sequenced in a targeted sequencing workflow.

Sequencing methodology of next-generation sequencing (NGS) platformstypically makes use of nucleic acid fragment libraries. In targetedsequencing techniques, a subset of fragments containing genes or regionsof interest of the genome are isolated from the nucleic acid library andsequenced. Targeted approaches using NGS allow researchers to focustime, expenses, and data analysis on specific areas of interest. Suchtargeted analysis can include the exome (the protein-coding portion ofthe genome), specific genes of interest (custom content), targets withingenes, or mitochondrial DNA. Targeted approaches contrast with wholegenome sequencing approaches that are more comprehensive, but that alsoinvolve sequencing regions of the genome that may not be of interest toall users.

In one example of a targeted sequencing technique, hybrid capturemethods use a panel or set of probes that hybridize to target sequencesin the nucleic acid library. Hybridization of the probes to the targetsequences allows these sequences to be separated from the rest of thefragments in the library for sequencing. By targeting only a portion ofthe nucleic acid library, hybrid capture methods avoid sequencing ofoff-target nucleic acid fragments that do not contain sequences ofinterest. However, unlike amplicon-based target enrichment methods,hybrid capture methods have a higher rate of off-target sequencing and,in turn, lower on-target specificity. For example, certain hybridcapture methods generally achieve only 40%˜60% efficiency, despite theuse of commercial hybridization blockers such as Cot1, tRNA, salmonsperm DNA, poly(dIdC) and blockers targeting the universal adapters oflibrary fragments. The off-target reads not only waste sequencing yield,but also potentially compromise variant calling for somatic mutations oflow frequency. Therefore, there is a need for improved enrichmentmethods that provide for higher specificity in targeted sequencingtechniques.

BRIEF SUMMARY

Presented herein are techniques for enrichment of target sequences in anucleic acid library and reducing the capture of off-target sequences bya set of target hybridization probes. Because target hybridizationprobes have imperfect specificity for their nucleic acid targets, asequencing run using a set of target hybridization probes may alsoinclude a certain percentage of reads that represent sequences that areoff-target. For example, in an exome sequencing reaction, certainhybridization probes may pull down intronic or intergenic sequences froma nucleic acid library along with target sequences. These off-targetfragments, once pulled down, are then present in the pool of nucleicacid fragments that are sequenced. While the sequencing informationrepresentative of the off-target reads is typically discarded, thepresent techniques use acquired sequencing information of theseoff-target reads to design hybridization probes that are specific forthe off-target sequences and that are used to separate and/or removefragments that include these sequences from the pool of fragmentscaptured by the target-specific hybridization probes. The off-targethybridization probes are designed based on analysis of the off-targetreads of a hybrid capture sequencing run that is performed with a set oftarget hybridization probes. In certain embodiments, the on-target probedesign may also be based on systematic off-target analysis acrosssamples to improve the specificity of the target hybridization probesfor their desired targets.

Presented herein is a method of reducing off-target capture in atargeted sequencing reaction. The method includes the steps of providinga set of off-target hybridization probes that specifically bind to aplurality of off-target sequences present in a nucleic acid librarygenerated from a sample, the nucleic acid library comprising a pluralityof nucleic acid fragments and providing a set of target-specifichybridization probes that specifically bind to a plurality of targetsequences present in the nucleic acid library. The method also includesthe steps of contacting the off-target hybridization probes with thenucleic acid library under conditions whereby the off-targethybridization probes hybridize to the off-target sequences andcontacting the target-specific hybridization probes with the nucleicacid library under conditions whereby the target-specific hybridizationprobes hybridize to the target sequences. The method also includes thesteps of selecting a group of nucleic acid fragments from the nucleicacid library bound to the target-specific hybridization probes; andsequencing the group of nucleic acid fragments bound to thetarget-specific hybridization probes.

Presented herein is also a method of providing probes for off-targetsequence capture in a targeted sequencing reaction. The method includesthe steps of receiving a request for a set of target-specifichybridization probes. The method also includes the steps of contactingthe target-specific hybridization probes with a reference nucleic acidlibrary generated from a reference sample, the nucleic acid librarycomprising a plurality of nucleic acid fragments, to generate areference group of target-specific and off-target nucleic acid fragmentsbound to the target-specific hybridization probes and separating thereference group of nucleic acid fragments bound to the target-specifichybridization probes from unbound nucleic acid fragments. The methodalso includes the steps of sequencing the reference group of nucleicacid fragments to generate reference sequencing data; identifyingoff-target sequences in the reference sequencing data; and providing aset of off-target hybridization probes based on the identifiedoff-target sequences.

Presented herein is also a sequencing kit for reducing off-targetcapture in a targeted sequencing reaction that includes a set ofoff-target hybridization probes that specifically bind to a plurality ofoff-target sequences present in a nucleic acid library generated from asample, the nucleic acid library comprising a plurality of nucleic acidfragments and a set of target-specific hybridization probes thatspecifically bind to a plurality of target sequences present in thenucleic acid library.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of target-specific hybridizationprobes binding to off-target fragments of a nucleic acid library;

FIG. 2 shows genome browser views off-target reads from six samples oftwo cell lines with reads distribution at a targeted region (left side)and an off-target region (right side) with similar coverage;

FIG. 3 is a graph showing genomic distribution of off-target reads;

FIG. 4 is a graph demonstrating similarity between target probes andoff-target peaks for a first sample;

FIG. 5 is a graph demonstrating similarity between target probes andoff-target peaks for a second sample;

FIG. 6 is a flow diagram of a method of providing off-targethybridization probes according to embodiments of the disclosure;

FIG. 7 is a flow diagram of a method of reducing off-target capture in atargeted sequencing reaction according to embodiments of the disclosure;

FIG. 8 is schematic representation of a competitive blocking method ofreducing off-target capture in a targeted sequencing reaction accordingto embodiments of the disclosure;

FIG. 9 is schematic representation of a pre-blocking method of reducingoff-target capture in a targeted sequencing reaction according toembodiments of the disclosure;

FIG. 10 is schematic representation of a pre-clearing method of reducingoff-target capture in a targeted sequencing reaction according toembodiments of the disclosure;

FIG. 11 is schematic representation of methods of reducing off-targetcapture in a targeted sequencing reaction used in conjunction withadaptor blockers according to embodiments of the disclosure;

FIG. 12 is schematic representation of methods of reducing off-targetcapture in a targeted sequencing reaction used in conjunction withattenuators according to embodiments of the disclosure;

FIG. 13 is schematic representation of fragments of a nucleic acidlibrary separated according to probe binding according to embodiments ofthe disclosure;

FIG. 14 is a graph demonstrating significant coverage drop on off-targetpeaks after pre-clearing, with each dot represents read coverage of anoff-target peak in control (x-axis) and in the same sample from thepre-clearing protocol (y-axis);

FIG. 15 is a graph demonstrating significant coverage drop on off-targetpeaks after pre-clearing, with each dot represents read coverage of anoff-target peak in control (x-axis) and in the same sample from thepre-clearing protocol (y-axis);

FIG. 16 is a graph demonstrating significant coverage drop on off-targetpeaks after pre-clearing, whereby each dot represents read coverage ofan off-target peak in control (x-axis) and in the same sample from thepre-clearing protocol (y-axis);

FIG. 17 is a graph demonstrating coverage gain and drop on on-targetregions, whereby dot represents read coverage of an on-target region incontrol (x-axis) and in the same sample with pre-cleaning (y-axis);

FIG. 18 is a graph demonstrating coverage gain and drop on on-targetregions, whereby dot represents read coverage of an on-target region incontrol (x-axis) and in the same sample with pre-cleaning (y-axis);

FIG. 19 is a graph demonstrating coverage gain and drop on on-targetregions, whereby dot represents read coverage of an on-target region incontrol (x-axis) and in the same sample with pre-cleaning (y-axis); and

FIG. 20 is a block diagram of a sequencing device that may be used inconjunction with the embodiments of the disclosure.

DETAILED DESCRIPTION

Hybrid capture methods in which target sequences are selected viabinding by hybridization probes are associated with a high off-targetbinding rate and low on-target specificity. The present techniquesimprove sequencing efficiency by reducing the presence of off-targetsequences in a hybrid capture sequencing workflow using a data-guidedapproach. While certain techniques may use blockers or bindingattenuators to influence probe binding, such approaches are notdata-guided. For example, salmon sperm DNA may be used to preventnon-specific binding of probes to reaction surfaces. However,nonspecific blockers do not prevent the binding of target-specificprobes to off-target sequences with similarity to target sequences.Target-specific probes have specificity for their intended targets.However, sequences present in off-target regions may be sufficientlysimilar to the target sequences (e.g., having short stretches ofhomology with the target, high string similarity) to permit at leastsome off-target binding of a target probe, albeit with lower specificityrelative to the target sequence binding. Off-target binding is moreprevalent in hybrid capture techniques relative to other targetedsequencing methodologies, in part because target-specific hybridizationprobes are typically longer oligonucleotides (80-120mer) relative to theprimers (25-30mer) in PCR-based methods, which may facilitate probebinding to off-target sequences having sufficient similarity to thetarget sequences. PCR-based targeted sequencing typically requires bothends of the primer binding to a specific area. The double binding needmakes random off-target binding slower to amplify compared to on-targetbinding, which in turn reduces off-target amplification. In anotherexample, longer oligonucleotides are statistically more likely thanshorter oligonucleotides to have contiguous base stretches within theoligonucleotides that are similar to the off-target sequences. Suchcomplementary or high similarity contiguous stretches may contribute tooff-target binding.

The present techniques use information about off-target sequences toimprove hybrid capture and decrease the percentage of off-targetcapture. A hybrid capture sequencing reaction may acquire sequence datafrom off-target sequences as a result of undesired off-target binding oftarget-specific hybridization probes. While such off-target sequencingdata is typically discarded, the present techniques harness thesequencing information of the off-target sequences for use in designingprobes specific for these off-target regions. Using probes with highspecificity for the off-target facilitates a reduction on the totalnumber of off-target regions present in a pool of sequenced fragments.As a result of the data-guided approach, the percentage of off-targetsequencing reads in a given sequencing run will be reduced. Accordingly,the present techniques provide the benefit of improving the efficiencyof a sequencing device by reducing the total amount of raw datagenerated in a sequencing run. Further, the reduction in off-targetreads present in the sequencing data also improves the efficiency ofdata analysis by reducing the amount of off-target sequence data to beidentified and excluded from analysis.

Turning to the figures, embodiments of the present techniques includeacquisition of off-target sequence data as an input for data-guideddesign of off-target hybridization probes. FIG. 1 is a schematicrepresentation of off-target binding by target-specific hybridizationprobes that leads to the acquisition of off-target sequence data.Referring first to FIG. 1, a schematic representation of a hybridcapture workflow with off-target binding by a target-specifichybridization probe is illustrated. Such off-target binding asillustrated may be used to acquire sequencing data that is used tocharacterize the off-target sequences as provided herein. In a targetedsequencing hybrid capture workflow, target-specific hybridization probesare designed to hybridize to sequences present in regions of interest ina sample. The workflow includes preparation of a nucleic acid library 10formed from a plurality of nucleic acid fragments 12 from the sample,such as a sample including genomic DNA (e.g., a human genome, an animalgenome, a bacterial genome) or other nucleic acids. The nucleic libraryincludes fragments having sequences from regions of interest (e.g.,fragment 12 a) that include target sequences 14 as well as fragmentsthat are off-target (e.g., fragment 12 b) having only off-targetsequences 16. It should be understood that fragments 12 that includetarget sequences 14 may be formed entirely from regions of interest ormay include other regions that are not of interest. The target-specifichybridization probes 20 are designed to be complementary to one or moretarget sequences 14 on a fragment 12. Accordingly, under hybridizationconditions, one or more target-specific hybridization probes 20 (e.g.,probes 20 a, 20 b) will bind to the complementary target sequences 14.This facilitates separation of fragments 12 that have the targetsequences 14 from the fragments 12 that do not have the target sequences14 (e.g., ones that have only off-target sequences 16) create atarget-enriched sample for sequencing.

As provided herein, a target sequence 14 is a nucleic acid sequencepresent in a nucleic acid library that is complementary to atarget-specific hybridization probe 20. Depending on the desiredsequencing outcome, the target sequences 14 may be exonic sequences forexome sequencing. Accordingly, in some embodiments, the target-specifichybridization probes 20 are directed to target sequences 14 of exons. Inanother embodiment, the target sequences 14 may be custom sequences, ordisease or allele-specific sequences. The target sequence 14 may be partof a region of interest in a nucleic acid sample, and thetarget-specific hybridization probe 20 may be designed based on variousmetrics to be specific for a portion of the region of interest.

As provided herein, a probe (e.g., a target-specific hybridization probe20) is an oligonucleotide, such as a single-stranded nucleic acidmolecule. The target-specific hybridization probe 20 may be part of aset or panel of target-specific hybridization probes 20. Thetarget-specific hybridization probes 20 may be 80-120 bases in length,80-100 bases in length, 90-110 bases in length, 100-120 bases in length,etc. In certain embodiments, if the target-specific hybridization probe20 is 80-120 bases in length, at least 30-50 of the bases of thetarget-specific hybridization probe are complementary to the targetsequence 14. It should be understood that a hybrid capture sequencingreaction may be performed using a set of target-specific hybridizationprobes 20, wherein different probes are representative of differenttarget sequences 14 in the nucleic acid library. For example, the set oftarget-specific hybridization probes 20 may be representative of atleast 2000 different target sequences 14, at least 5000 different targetsequences 14, at least 10,000 different target sequences 14, and so on.Further, while the disclosed embodiments are discussed with regard tohybrid capture technologies, incorporation of the techniques providedherein may also be implemented with PCR or amplicon-based sequencingtechniques. In such embodiments, the target-specific hybridizationprobes 20 may be on the order of 20-40 bases in length.

In certain embodiments, the target-specific hybridization probes 20 mayhave modifications that facilitate separation of bound fragments 12 fromthe unbound fragments 12. Such modifications may include biotinylationof the probe to facilitate selection via streptavidin (e.g.,streptavidin beads). However, it should be understood that the probes asprovided herein may be coupled to other an affinity binding moleculethat is part of a binding pair. For example, biotin and streptavidin,biotin and avidin, or digoxigenin and a specific antibody that bindsdigoxigenin are examples of specific binding pairs. The affinity bindingmolecule may be an antibody ligand capable of being conjugated to anucleotide. In certain embodiments, the modification is provided at the5′ or the 3′ end of the probe. Further, in other embodiments, the probesmay be unmodified. The target-specific hybridization probes 20 may alsoinclude unique barcodes or sequences that facilitate identification.Such sequences may part of a region of the probe 20 that isnon-complementary to the target sequence 14. The target-specifichybridization probes 20 may be in solution or immobilized on a solidsupport (e.g., an array).

As shown in FIG. 1, fragment 12 b includes an off-target sequence 16 towhich a target-specific hybridization probe 20 c is bound in an exampleof off-target binding. Once bound to the target-specific hybridizationprobe 20 c, the off-target fragment 12 b is separated from the rest ofthe nucleic acid library 10 along with the fragments 12 that includetarget sequences 14 and processed through the sequencing reaction.Accordingly, the off-target sequence read from the fragment 12 b will beincluded in the sequencing data. While the probe 20 c has higherspecificity for its target sequence 14 relative to the off-targetsequence 16, the binding conditions nonetheless permit binding of thesome of the target-specific hybridization probe 20 c to the off-targetsequence 16. In other words, certain off-target sequences 16 may sharesufficient similarity to the target sequence 14 for the probe 20 c topermit the binding to occur.

As provided herein, an off-target sequence 16 is a sequence that is notan intended target of one or more of the target-specific hybridizationprobes 20. In one example, if the target-specific hybridization probes20 are for exome sequencing, an off-target sequence 16 may be anintronic or intergenic sequence. In certain embodiments, atarget-specific hybridization probe 20 is capable of binding to anoff-target sequence 16 with lower specificity than for the intendedtarget sequence.

An examination of off-target sequences was performed to demonstrate thatthe off-target sequences are relatively stable between samples. FIG. 2shows sequencing results for systematic off-target regions that werehighly reproducible across different samples and replicates from 6samples of two cell lines as shown in genome browser views of the readsdistribution at (a) a targeted region and (b) an off-target region withsimilar coverage. Each track is one sample. FIG. 2 demonstrates thatoff-target binding tends to be stable and that the incidence,distribution and representation of the off-targets were systematicrather than random.

FIG. 3 is a graph showing genomic distribution of off-target reads.Off-target regions showed similar coverage across different replicates.In the samples studied, 45% of the off-target sequences were located inintrons, 48% in intergenic regions, and 7% in exons.

The sequence similarities between the off-target regions and captureprobes also indicate that off-target reads were likely pulled down byprobes, rather than by random binding. FIGS. 4-5 show graphsdemonstrating similarity between target probes and off-target peaks.Systematic off-target regions had sequence similarity with target probesshown in different samples. The 10 kb downstream regions were subject tothe same analysis as negative control. Each dot represents the number ofprobes sharing sequence similarity with an off-target peak (x-axis) andwith its 10 kb downstream region (y-axis). Accordingly, the data showthat the off-target sequences are likely to be specific and reproduciblebetween samples for a given set of target-specific hybridization probes.From the sequencing data it was observed that there are off-targetregions with coverage significantly higher than general background andcomparable to or even higher than actual targets. If ranked bycontribution to coverage, a portion of off-target loci contribute to themajority of off-target reads. If fragments of these regions can bereduced during sequencing, coverage on actual target can be improvedconsequently. To that end, the present techniques include off-targethybridization probes that are specific for the off-target sequences thatare stable and/or prevalent between samples. Further, in certainembodiments, off-target hybridization probes may be designed for theoff-target loci that contribute to the majority of off-target reads.

FIG. 6 is a flow diagram of a method 30 for identifying off-targetsequences as provided herein. Targeted sequencing (e.g., hybrid capturesequencing) using a set of target-specific hybridization probes isperformed (step 32) and sequencing data acquired (step 34 to identifyloci of high-frequency off-target reads in the hybrid capture sequencingdata (step 36). Based on the loci of the off-target reads, off-targethybridization probes are designed (step 38) and provided for use inconjunction with the target-specific hybridization probes (step 40).

As provided herein, sequencing data may include raw data as well as basecall data for the sequenced fragments of the nucleic acid library.Further, the sequencing data may have undergone alignment and assemblyso that the genome loci of the assembled fragments can be identified.Accordingly, the sequence data may include sequence information andlocation information for the assembled fragments such that off-targetdata is identifiable based at least in part on the location of thesequenced fragments. In addition, the sequencing data may includecoverage data of off-target sequence reads so that the off-targetprevalence as well as locations may be assessed. In this manner, thehighest prevalence sequence reads (i.e., highest coverage) for variousoff-target loci may be identified. In certain embodiments, theoff-target reads are ranked according to coverage to identify thehighest frequency off-target loci. The off-target hybridization probesmay be designed based on the highest 50, 100, 1000, or 2000 loci. In oneembodiment, the design is based on a user-specified number of the rankedsequences.

In one embodiment, the method 30 may be performed as part of a workflowfor generating a panel of target-specific hybridization probes. Based ona request for a particular panel of target-specific hybridizationprobes, the method 30 is initiated on a reference sample to identify andassess the off-target sequences. The reference sample may be an internalstandard that is known to be a high quality sample. In anotherembodiment, the method 30 is initiated upon receipt of a customerrequest for a custom panel of target-specific hybridization probes. Aspart of synthesizing the custom panel, the method 30 is performed toidentify potential off-target sequences. Accordingly, the method 30 maybe performed in response to a user or customer input.

Based on the identified off-target sequences, a set of off-targethybridization probes may be identified and synthesized to be provided aspart of a sequencing kit. The off-target hybridization probes may be anoptional add-on item to improve sequencing yield and reduce off-targetsequence capture. In another embodiment, the method 30 may also includegenerating an estimate of sequencing cost reduction for the referencesample based on an estimated reduction in off-target sequencing reads.For example, if a typical hybrid capture sequencing run generates 60%target reads and 40% off-target reads, then 40% of the cost ofsequencing is attributable to off-target sequences. If the set ofoff-target hybridization probes is designed to correspond to off-targetsequences that represent about 50% of the off-target coverage in thereference sequencing data, then the off-target hybridization probes arecapable of reducing off-target reads by 50%. Accordingly, a sequencingrun using the off-target hybridization probes to reduce off-targetcapture may be estimated to lower costs by 20% relative to the control.In this manner, a user may determine if the cost of the off-targethybridization probes will generate sufficient savings on sequencing. Themethod 30 may also permit dynamic estimates based on variable userinputs. For example, reducing the total number of off-target sequencesof the off-target hybridization probes will reduce probe cost, but maybe associated with a slight increase in off-target sequence capture,resulting in an associated rise in estimated sequencing costs relativeto a selection of a higher number of off-target sequences of theoff-target hybridization probes. In another embodiment, the user mayprovide a total sequencing budget, including any target and off-targetprobe costs, for a given sample, and a determination may be made if costsavings can be achieved using the off-target hybridization probes.

As provided herein, an off-target hybridization probe (e.g., off-targethybridization probe 60, see FIGS. 8-13) has higher specificity for anoff-target sequence (e.g., off-target sequence 16, see FIG. 1) relativeto any target sequences. An off-target hybridization probe is anoligonucleotide, such as a single-stranded nucleic acid molecule. Theoff-target hybridization probe may be part of a set of off-targethybridization probes. The off-target hybridization probes may be 80-120bases in length, 80-100 bases in length, 90-110 bases in length, 100-120bases in length, etc. In certain embodiments, if the off-targethybridization probe is 80-120 bases in length, at least 30-50 of thebases of the off-target hybridization probe are complementary to theoff-target sequence. Further, while the disclosed embodiments arediscussed with regard to hybrid capture technologies, incorporation ofthe techniques provided herein may also be implemented with PCR oramplicon-based sequencing techniques. In such embodiments, theoff-target hybridization probes 20 may be on the order of 20-40 bases inlength. In one embodiment, the off-target hybridization probes may beabout the same length as the target specific hybridization probes. Inanother embodiment, the off-target hybridization probes may be adifferent length (e.g., shorter or longer) relative to the targetspecific hybridization probes.

It should be understood that a targeted sequencing reaction may beperformed using a set of target-specific hybridization probes 20together with (e.g., in parallel or in sequence) off-targethybridization probes, wherein off-target hybridization probes arerepresentative of different off-target sequences in the nucleic acidlibrary. For example, the set of off-target hybridization probes may berepresentative of at least 50 different off-target sequences, at least100 different off-target sequences, at least 10000 different off-targetsequences, and so on. In another embodiment, a set of target sequencesrepresents a greater number of different sequences than a set ofoff-target sequences for the probes used in a hybrid capture sequencingas provided herein. For example, a ratio of the number of differenttarget sequences in the target-specific hybridization probes to thenumber of different off-target sequences in the off-target hybridizationprobes may be 2:1, 3:1, 4:1, 5:1 or greater in certain embodiments.There are certain advantages to providing a limited number of off-targethybridization probes due to the cost of manufacturing additional probesfor use. Accordingly, the ranking of the prevalence of off-targetsequences may be used to permit user selection of a number of desiredoff-target hybridization probes. Further, certain highly prevalentoff-target sequences may be present in the total pool of off-targetsequences to such a high degree that having a limited number ofoff-target hybridization probes specific for highly prevalentoff-targets may nonetheless yield a high reduction in off-targetsequence capture.

In certain embodiments, the off-target hybridization probes may havemodifications that facilitate separation of bound fragments from theunbound fragments. Such modifications may include biotinylation of theprobe to facilitate selection via streptavidin (e.g., streptavidinbeads). However, it should be understood that the probes as providedherein may be coupled to other an affinity binding molecule that is partof a binding pair. For example, biotin and streptavidin, biotin andavidin, or digoxigenin and a specific antibody that binds digoxigeninare examples of specific binding pairs. In certain embodiments, themodification is provided at the 5′ or the 3′ end of the probe. Further,in other embodiments, the probes may be unmodified.

The off-target hybridization probes may also include unique barcodes orsequences that facilitate identification. Such sequences may part of aregion of the probe that is non-complementary to the off-targetsequences. The off-target hybridization probes may be in solution orimmobilized on a solid support (e.g., an array). In another embodiment,the target-specific hybridization probes and the off-targethybridization probes are provided as similar length probes, i.e., allwithin a certain range. Accordingly, in a specific embodiment, thetarget-specific hybridization probes and the off-target hybridizationprobes are all in a range of 80-120 bases in length. In anotherembodiment, the target-specific hybridization probes and the off-targethybridization probes are all in a range of 20-40 bases in length. In yetanother embodiment, the target-specific hybridization probes have alength all in a first range and the off-target hybridization probes havea length all in a second range, whereby the first range and the secondrange are different. In one embodiment, the first range encompasseslonger probe lengths than the second range. In another embodiment, thefirst range encompasses shorter probe lengths than the second range.

In certain embodiments of the disclosure, providing the off-targethybridization probes comprises providing the off-target hybridizationprobes as part of a sequencing kit for use with the target-specifichybridization probes. The off-target hybridization probes may bespecific for only certain types of off-target sequences (e.g., introns,intergenic regions). In this manner, a user may select the off-targetsequences of interest. In another embodiment, providing the off-targethybridization probes comprises providing the off-target hybridizationprobes as part of a request or order for a custom target-specifichybridization probe panel. When the request for the custom panel isreceived, the synthesis facility may also perform the steps of themethod 30 to determine the off-target sequences of concern (e.g., highlyprevalent off-target sequences) for the custom panel and provideoff-target hybridization probes to reduce off-target reads from theseidentified off-target sequences.

In another embodiment, a universal set of off-target hybridizationprobes may be provided. That is, regardless of the particular panel oftarget-specific hybridization probes used, certain off-target reads maybe common across a species. In one implementation, a species-specificset of off-target hybridization probes may be used to de-host a sample,such as in microbiology, infectious disease, food safety, and qualitymonitoring. A universal set and/or a species-specific set may bedetermined using the data-guided techniques as provided herein. Forexample, the universal set or the species-specific set may be selectedby performing sequencing on reference samples using different panels oftarget-specific hybridization probes (e.g., using a plurality ofhuman-specific panels or using a plurality of cancer-specific panels)and selecting the top-ranked (i.e., most prevalent) off-target sequencesfrom the sequencing data from all of the different panels to design theoff-target hybridization probes. In one embodiment, the top-ranked setmay include only the off-target sequences that are common betweensamples sequenced using different panels. In another embodiment, thetop-ranked set may be representative of a pool of all of the off-targetsequences in the sequencing data using the different panels, such thatsome sequences in the pool are only off-target for a given panel.However, the top-ranked set will nonetheless include a number ofoff-target reads represented in the sequencing data for each sample suchthat the universal set, when used, will reduce off-target capture whenused in conjunction with any of the panels.

Also provided herein are methods of implementing targeted sequencingusing the off-target hybridization probes as provided herein. FIG. 7 isa method 50 of reducing off-target capture in a targeted sequencingreaction. The nucleic acid library may be prepared (step 52) accordingto a desired technique to facilitate fragment formation of theappropriate size. The library, once prepared, is contacted withoff-target hybridization probes and target-specific hybridization probes(step 54), either in sequence or in parallel, under conditions thatpermit probe binding. For example, double-stranded fragments may bedenatured to yield single-stranded fragments. The fragments bound to thetarget-specific hybridization probes are selected for sequencing (step56), e.g., via the techniques disclosed in FIGS. 8-10. In oneembodiment, the selecting is accomplished by separating fragments boundto the target-specific hybridization probes from fragments bound tooff-target hybridization probes as well as from fragments not bound toany probes. In another embodiment, the selecting is accomplished byfirst removing fragments bound to off-target hybridization probes andthen removing fragments not bound to any probes. Once selected, thefragments bound to the target-specific hybridization probes aresequenced (step 58) to yield sequencing data with a reduction in offtarget sequence reads relative to a control. For example, a control ofthe sample that is contacted with only target-specific hybridizationprobes and not with off-target hybridization probes.

FIG. 8 is a schematic illustration of an implementation of the method 50using competitive blocking. In the illustrated embodiment, off-targethybridization probes 60 are synthesized as 5′ un-modified (withoutbiotin) and used in a hybridization reaction along with (i.e., at thesame time as or in the same solution) target-specific hybridizationprobes 20 with a biotinylated end 62. The unmodified off-targethybridization probes 60 compete for binding to the off-target loci withthe target-specific hybridization probes 20. This in turn makes theoff-target loci inaccessible or less accessible to the target-specifichybridization probes 20. Separation of the target-specific hybridizationprobes 20 bound to the fragments 12 may be accomplished usingstreptavidin, which binds to the biotinylated ends 62 and not to theoff-target hybridization probes 60. The resulting group of separatedtarget-specific hybridization probes 20 bound to the fragments 12 isenriched for target sequences 14 and has a reduction in off-targetsequences 16 relative to a control group in which no off-targethybridization probes 60 are used.

FIG. 9 is a schematic illustration of an implementation of the method 50using pre-blocking. In the illustrated embodiment, unmodified off-targethybridization probes 60 are first contacted with the library topre-block the corresponding off-target sequences 16. The target-specifichybridization probes 20 are contacted with the library after thepre-blocking has occurred, i.e., only after the off-target hybridizationprobes 60 are bound. The off-target hybridization probes 60 aresynthesized as 5′ un-modified (without biotin) while the target-specifichybridization probes 20 have biotinylated ends 62. Separation of thetarget-specific hybridization probes 20 bound to the fragments 12 may beaccomplished using streptavidin, which binds to the biotinylated ends 62and not to the off-target hybridization probes 60. The resulting groupof separated target-specific hybridization probes 20 bound to thefragments 12 is enriched for target sequences 14 and has a reduction inoff-target 16 sequences relative to a control group in which nooff-target hybridization probes 60 are used.

FIG. 10 is a schematic illustration of an implementation of the method50 using pre-clearing. In the illustrated embodiment, modifiedoff-target hybridization probes 60, shown with biotinylated ends 68, arefirst contacted with the library and permitted to bind withcorresponding off-target sequences 16. The off-target hybridizationprobes 60 bound to the fragments that include off-target sequences 16are pulled out using with streptavidin beads to pre-clear, i.e., pullthe undesirable off-target loci out of the reaction. The pre-clearedsupernatant is retained for the subsequent hybrid capture reaction withthe modified target-specific hybridization probes 20 having biotinylatedends 62. The target sequences 14 remaining that are bound to thetarget-specific hybridization probes 20 are separated from any unboundfragments and then sequenced. The resulting group of pre-cleared andseparated target-specific hybridization probes 20 bound to the fragments12 is enriched for target sequences 14 and has a reduction in off-target16 sequences relative to a control group in which no off-targethybridization probes 60 are used.

It should be understood that the target-specific hybridization probes 20and the off-target hybridization probes 60 as provided herein may beused in conjunction with blockers or other approaches used in hybridcapture to reduce probe self-annealing, sticky probes, or nonspecificbinding. FIGS. 11-12 illustrate examples of approaches that may becombined, either in parallel or in sequence, with the off-targethybridization probes 60 and the embodiments disclosed herein forimproved results. These examples are merely illustrative, and are notintended to be limiting. FIG. 11 shows an example of antisense adapterblockers 70 that prevent adapters 72 from self-annealing. FIG. 12 showsan example of one or more probe attenuators 76 that are antisense fortarget-specific hybridization probes 20 to a “hyper” or sticky targetsequence 14.

In one embodiment, the off-target hybridization probes are specific forthe highly enriched off-target regions to provide reduction of themost-prevalent off-target reads. Where an off-target sequence has ahighly similar sequence to the actual target region, use of anoff-target hybridization probe specific for that highly similar sequencecould cause an unintended coverage drop for the target region having thesimilar sequence. To prevent this from happening, in one embodiment,off-target hybridization probes may be selected only from off-targetregions having less than a threshold similarity with a target sequenceaccording to one or more similarity metrics (e.g., Damerau-Levenshteindistance, Needleman-Wunsch algorithm, BLAST score). In one embodiment, athreshold percent identity or identity score is used to qualifyoff-target hybridization probes, with only off-target sequences havingless than a predetermined percent identity (e.g., less than 50%, lessthan 25%) with a target sequence being qualified. For example, in oneembodiment, only off-target sequences that do not contain matches of 15or more contiguous bases with a target sequence will be qualified foroff-target hybridization probe design. Those off-target sequences with15 or more contiguous bases in common with a target sequence are notused as the basis for any off-target hybridization probes, even if suchoff-target sequences are highly prevalent. In another example, becausethe loci of off-target sequences are known, the sequence for which theoff-target hybridization probe is specific can be shifted 5′ or 3′ awayfrom the highly similar region, e.g., moved 20-50 bases 5′ or 3′ suchthat the targeted region has a lower similarity score.

As shown in FIG. 13, the nucleic acid library used in a hybrid capturesequencing as provided herein is contacted with target-specifichybridization probes 20 and off-target hybridization probes 60, eitherin sequence or in parallel. The contacting yields a target group 80 offragments 12 including target sequences 14 bound to the target-specifichybridization probes 20, an off-target group 82 of fragments 12including off-target sequences bound to the off-target hybridizationprobes 60, and an unbound group 84 of fragments 12 that are not bound toany probes. The unbound group 84 includes sequences for which notarget-specific hybridization probes 20 or off-target hybridizationprobes 60 were designed. Selection of the target group 80 for sequencingand not the off-target group 82 and/or the unbound group 84 may beaccomplished via the techniques disclosed herein. For example,target-specific hybridization probes 20 that include a biotinmodification may be selected by binding with avidin or streptavidincoupled to beads or a support. Where the off-target hybridization probes60 are unmodified, the off-target group and the unbound group 84 can beremoved by washing.

In another embodiment, it may be desirable to retain the off-targetgroup 82 to assess probe quality. The pre-clearing technique (see FIG.10) may be used to pull out the fragments 12 bound to the off-targethybridization probes 60, i.e., the off-target group 82. These fragments12 in the off-target group 82 may be sequenced and evaluated for thepresence of any target sequences 14. Any target sequences present in theoff-target group 82 may then be assessed for similarity to one or moreoff-target hybridization probes, which in turn may be removed from theset or redesigned.

In one example, to find the consistent off-target regions, a set ofrepresentative samples, e.g. a set of samples of different celllines/tissues sequenced with good quality, were selected. Firstly,on-target reads were filtered out of the sequencing data, then regionshighly enriched for off-target reads were called using peak-callingtools GEM for the ENCODE project. However, other peaking callingalgorithms may also be used. Overlapping peaks from different sampleswere then extracted and peaks within 50 bp were merged and only thosethat are 400 bp or more away from the targets were kept. The off-targetpeaks that were identified previously were sorted by the averagecoverage. According to the ranking those with significantly highcoverage were choose to design reducers against. Off-targethybridization probes were designed to be specific for off-target regionsthat contributed to about 50% of the total off-target reads.DesignStudio (Illumina Inc.) was be utilized to design the off-targethybridization probes, representative of approximately 2000 off-targetsequences.

Using off-target probe design as outlined herein, FIGS. 14-16 showgraphs demonstrating significant coverage drop on off-target peaks afterpre-clearing. Each dot represents read coverage of an off-target peak incontrol (x-axis) and in the same sample from the pre-clearing protocol(y-axis). The box 100 in FIG. 14 highlights the significant differencein the read coverage of the control vs. the pre-clearing protocol.

FIGS. 17-19 show graphs demonstrating coverage gain and drop onon-target regions. Each dot represents read coverage of an on-targetregion in control (x-axis) and in the same sample with pre-cleaning(y-axis). The area 104 highlights the coverage gain whereas the box 102underlines coverage drop as shown in FIG. 17.

The techniques provided herein address the problem of a high off-targetcapture rate by using guided information from data analysis on theoff-target regions. Prior attempts to solve this issue have utilizedCot1, tRNA, poly(dI-dC), adapter blockers and blockers forhigh-representation genes (e.g. anti-mitochondrial gene blockers). Incontrast to those methodologies, the methods presented herein representthe first data driven approach. Furthermore, using off-targethybridization probes to clean or remove the unwanted DNA fragments outof sample libraries prior to target-specific binding is a novelapproach. Further, the identified systematic off-target regions that arestable between samples as well as different sets or panels ofhybridization probes may not necessarily be identified by theconventional wisdom. For example, they may not necessarily beidentifiable repetitive elements such as Alu, SINE, LINE, or etc. Insome embodiments, the approach described herein can be applied to othergenomes to develop specie-specific off-target hybridization probes formetagenomic applications or contamination elimination in sample prep.

The techniques disclosed herein may be implemented in conjunction with asequencing device and/or a sequence analysis device. FIG. 20 is aschematic diagram of a sequencing device 120 that may be used, forexample, to acquire and assess off-target reads that may be used in turnto design off-target hybridization probes. In another example, thesequencing device 120 may be used to acquire and assess sequencing dataafter using the off-target hybridization probes. The acquired sequencingdata will have reduced off-target reads relative to a control that wasnot contacted with the off-target hybridization probes. The sequencingdevice 120 may be implemented according to any sequencing technique,such as those incorporating sequencing-by-synthesis methods described inU.S. Patent Publication Nos. 2007/0166705; 2006/0188901; 2006/0240439;2006/0281109; 2005/0100900; U.S. Pat. No. 7,057,026; WO 05/065814; WO06/064199; WO 07/010,251, the disclosures of which are incorporatedherein by reference in their entireties. Alternatively, sequencing byligation techniques may be used in the sequencing device 120. Suchtechniques use DNA ligase to incorporate oligonucleotides and identifythe incorporation of such oligonucleotides and are described in U.S.Pat. Nos. 6,969,488; 6,172,218; and 6,306,597; the disclosures of whichare incorporated herein by reference in their entireties. Someembodiments can utilize nanopore sequencing, whereby target nucleic acidstrands, or nucleotides exonucleolytically removed from target nucleicacids, pass through a nanopore. As the target nucleic acids ornucleotides pass through the nanopore, each type of base can beidentified by measuring fluctuations in the electrical conductance ofthe pore (U.S. Pat. No. 7,001,792; Soni & Meller, Clin. Chem. 53,1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); and Cockroft, etal. J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which areincorporated herein by reference in their entireties). Yet otherembodiments include detection of a proton released upon incorporation ofa nucleotide into an extension product. For example, sequencing based ondetection of released protons can use an electrical detector andassociated techniques that are commercially available from Ion Torrent(Guilford, Conn., a Life Technologies subsidiary) or sequencing methodsand systems described in US 2009/0026082 A1; US 2009/0127589 A1; US2010/0137143 A1; or US 2010/0282617 A1, each of which is incorporatedherein by reference in its entirety. Particular embodiments can utilizemethods involving the real-time monitoring of DNA polymerase activity.Nucleotide incorporations can be detected through fluorescence resonanceenergy transfer (FRET) interactions between a fluorophore-bearingpolymerase and γ-phosphate-labeled nucleotides, or with zeromodewaveguides as described, for example, in Levene et al. Science 299,682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008);Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), thedisclosures of which are incorporated herein by reference in theirentireties. Other suitable alternative techniques include, for example,fluorescent in situ sequencing (FISSEQ), and Massively ParallelSignature Sequencing (MPSS). In particular embodiments, the sequencingdevice 120 may be a HiSeq, MiSeq, or HiScanSQ from Illumina (La Jolla,Calif.).

In the depicted embodiment, the sequencing device 120 includes aseparate sample processing device 122 and an associated sequenceanalysis device 124. Further, it is contemplated that the sequenceanalysis device 124 may be implemented separately form and notassociated with the sample processing device 122. Accordingly, in suchan embodiment, sequence analysis device 124 receives data from a remotesample processing device 122. However, these may be implemented as asingle device. Further, the associated sequence analysis device 124 maybe local to or networked with the sample processing device 122. In thedepicted embodiment, the biological sample may be loaded into the sampleprocessing device 122 as a sample slide 126 that is imaged to generatesequence data. For example, reagents that interact with the biologicalsample fluoresce at particular wavelengths in response to an excitationbeam generated by an imaging module 128 and thereby return radiation forimaging. For instance, the fluorescent components may be generated byfluorescently tagged nucleic acids that hybridize to complementarymolecules of the components or to fluorescently tagged nucleotides thatare incorporated into an oligonucleotide using a polymerase. As will beappreciated by those skilled in the art, the wavelength at which thedyes of the sample are excited and the wavelength at which theyfluoresce will depend upon the absorption and emission spectra of thespecific dyes. Such returned radiation may propagate back through thedirecting optics. This retrobeam may generally be directed towarddetection optics of the imaging module 128.

The imaging module detection optics may be based upon any suitabletechnology, and may be, for example, a charged coupled device (CCD)sensor that generates pixilated image data based upon photons impactinglocations in the device. However, it will be understood that any of avariety of other detectors may also be used including, but not limitedto, a detector array configured for time delay integration (TDI)operation, a complementary metal oxide semiconductor (CMOS) detector, anavalanche photodiode (APD) detector, a Geiger-mode photon counter, orany other suitable detector. TDI mode detection can be coupled with linescanning as described in U.S. Pat. No. 7,329,860, which is incorporatedherein by reference. Other useful detectors are described, for example,in the references provided previously herein in the context of variousnucleic acid sequencing methodologies.

The imaging module 128 may be under processor control, e.g., via aprocessor 130, and the sample preparation device 122 may also includeI/O controls 132, an internal bus 134, non-volatile memory 136, RAM 138and any other memory structure such that the memory is capable ofstoring executable instructions, and other suitable hardware componentsthat may be similar to those described with regard to FIG. 20. Further,the associated sequence analysis device 124 may also include a processor140, I/O controls 144, a communications module 152, and a memoryarchitecture including RAM 146 and non-volatile memory 148, such thatthe memory architecture is capable of storing executable instructions150. The hardware components may be linked by an internal bus 153, whichmay also link to the display 154. In embodiments in which the sequencingdevice 122 is implemented as an all-in-one device, certain redundanthardware elements may be eliminated.

The sequencing device 120 may be used to request target-specifichybridization probes. Further, the sequencing device 120 may be used toprovide user inputs for off-target hybridization probe preparation. Theuser may provide inputs specifying a desired number of highest rankedsequences to be prepared as the set of off-target hybridization probes.The selections may alternatively or additionally be based on a desiredpercentage of off-target reduction.

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosure of these publicationsin their entireties is hereby incorporated by reference in thisapplication. The term comprising is intended herein to be open-ended,including not only the recited elements, but further encompassing anyadditional elements. While only certain features of the invention havebeen illustrated and described herein, many modifications and changeswill occur to those skilled in the art. It is, therefore, to beunderstood that the appended claims are intended to cover all suchmodifications and changes as fall within the true spirit of theinvention. Further, elements of the disclosed embodiments may becombined or exchanged. Accordingly, other embodiments are within thescope of the following claims.

What is claimed is:
 1. A method of reducing off-target capture in atargeted sequencing reaction, comprising: providing off-targethybridization probes that specifically bind to a plurality of off-targetsequences present in a nucleic acid library generated from a sample, thenucleic acid library comprising a plurality of nucleic acid fragments;providing target-specific hybridization probes that specifically bind toa plurality of target sequences present in the nucleic acid library,wherein the target specific hybridization probes comprise a set ofprobes; contacting the off-target hybridization probes with the nucleicacid library under conditions whereby the off-target hybridizationprobes hybridize to the off-target sequences, wherein the off-targethybridization probes are specific for off-target sequences identified byassessing off-target sequence data present in reference sequence dataacquired from a reference nucleic acid library contacted with thetarget-specific hybridization probes, wherein the target specifichybridization probes comprise the set of probes; contacting thetarget-specific hybridization probes with the nucleic acid library underconditions whereby the target-specific hybridization probes hybridize tothe target sequences; selecting a group of nucleic acid fragments fromthe nucleic acid library bound to the target-specific hybridizationprobes; and sequencing the group of nucleic acid fragments bound to thetarget-specific hybridization probes.
 2. The method of claim 1, whereina subset of nucleic acid fragments comprise the off-target sequences,and wherein the method further comprises not sequencing the subset ofnucleic acid fragments bound to the off-target hybridization probes. 3.The method of claim 1, comprising sequencing the subset of nucleic acidfragments after the selecting and providing sequencing data of thesubset to determine a quality metric for one or both of thetarget-specific hybridization probes or the off-target hybridizationprobes.
 4. The method of claim 1, wherein the nucleic acid librarycomprises genomic DNA fragments and the off-target hybridization probesare specific for genomic sequences that are identified as off-targetsequences.
 5. The method of claim 4, wherein the genomic DNA fragmentsare derived from human genomic DNA.
 6. The method of claim 1, whereinone or both of contacting the off-target hybridization probes with thenucleic acid library or contacting the target-specific hybridizationprobes with the nucleic acid library are performed in the presence ofglobal hybridization blockers.
 7. The method of claim 6, wherein theglobal hybridization blockers comprise one or more of Cot1 DNA, tRNA,salmon sperm DNA, poly(dIdC) and blockers targeting universal adaptersof library fragments.
 8. The method of claim 1, wherein contacting theoff-target hybridization probes with the nucleic acid library andcontacting the target-specific hybridization probes with the nucleicacid library are performed at the same time.
 9. The method of claim 1,wherein contacting the off-target hybridization probes with the nucleicacid library is performed before contacting the target-specifichybridization probes with the nucleic acid library.
 10. The method ofclaim 9, wherein selecting the group of nucleic acid fragments from thenucleic acid library bound to the target-specific hybridization probescomprises removing nucleic acid fragments bound to the off-targethybridization probes before contacting the target-specific hybridizationprobes with the nucleic acid library.
 11. The method of claim 1, whereinthe off-target hybridization probes are 5′ un-modified and thetarget-specific hybridization probes are 5′ biotinylated.
 12. The methodof claim 1, wherein the off-target hybridization probes and thetarget-specific hybridization probes are 5′ biotinylated.
 13. The methodof claim 1, wherein selecting the group comprises using streptavidinbeads that bind to one or both of the off-target hybridization probes orthe target-specific hybridization probes.
 14. The method of claim 1,wherein the target-specific hybridization probes are specific for exonicsequences and the off-target hybridization probes are specific for oneor both of intronic sequences or intergenic sequences.
 15. The method ofclaim 1, wherein the target-specific hybridization probes comprise aplurality of probes specific for a first number of different targetsequences, wherein the off-target hybridization probes comprise aplurality of probes specific for a second number of different off-targethybridization sequences, and wherein the first number is greater thanthe second number.
 16. The method of claim 15, wherein the second numberis determined via a user-specified setting.
 17. The method of claim 15,wherein the second number is determined based on an estimated percentagereduction of off-target sequence reads in the sequencing data relativeto a control, wherein the estimated percentage reduction is determinedbased on a prevalence of off-target sequence reads corresponding to theoff-target hybridization probes in the off-target sequence data.
 18. Themethod of claim 1, wherein the off-target hybridization probes and thetarget-specific hybridization probes are single-stranded nucleic acidshaving lengths a range of 80 to 120 bases.
 19. The method of claim 1,wherein providing the off-target hybridization probes based on theidentified off-target sequences comprises ranking a prevalence of aplurality of off-target sequences in the sequencing data and selecting aplurality of highest prevalence off-target sequences to design theoff-target hybridization probes such that the off-target hybridizationprobes are specific for the highest prevalence off-target sequences. 20.The method of claim 1, wherein providing the off-target hybridizationprobes based on the identified off-target sequences comprises selectinga subset of the identified off-target sequences having less than athreshold similarity with the target sequences to design the off-targethybridization probes.
 21. The method of claim 1, wherein thetarget-specific hybridization probes are provided based on a user inputand wherein the off-target hybridization probes are designed afterreceiving the user input.
 22. The method of claim 21, wherein the userinput defines a custom set of the target-specific hybridization probes.23. The method of claim 1, comprising receiving an estimated reductionin sequencing cost associated with use of the off-target hybridizationprobes.
 24. The method of claim 1, wherein the reference nucleic acidlibrary is contacted with the target-specific hybridization probes andnot the off-target hybridization probes to acquire the referencesequence data.
 25. The method of claim 1, wherein the reference nucleicacid library is generated from a reference sample and wherein thenucleic acid library is generated from a user sample.